In vitro culture studies on Hemigraphis alternata (Burm.f.) T. Ander. (Acanthaceae): A Potential medicinal herb

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1 WWJMRD 2017; 3(7): Impact Factor MJIF: 4.25 e-issn: Delse P. Sebastian Satheesh George Binu Thomas Correspondence: Binu Thomas In vitro culture studies on Hemigraphis alternata (Burm.f.) T. Ander. (Acanthaceae): A Potential medicinal herb Delse P. Sebastian, Satheesh George, Binu Thomas Abstract The current situation of medicinal plants and increasing demand of plant derived drugs suggest an immediate need to conserve our medicinal plant resources. The present study attempted to develop in vitro micropropagation protocol for the important medicinal plant Hemigraphis alternata (Burm.f.) T. Ander. From this experiment it is clear that higher amount of cytokinins and lower auxin induce shoot regeneration and high amount of auxin and lower amount of cytokinins induce root regeneration. Similarly the most effective shoot regeneration was found in the M.S. medium supplemented with KIN 2 mg/l. It shows an effective rooting in the ¼ strength M.S. medium supplemented with NAA 0.5 mg/l. large numbers of multiple shoots were also formed in the M.S. medium is supplemented with KIN 2 mg/l. Keywords: In vitro culture, Hemigraphis alternata, Acanthaceae, medicinal herb Introduction Plant tissue culture is a technique of culturing plant cells, tissues and organs on synthetic media under aseptic environment and controlled conditions of light, temperature and humidity. The development of plant tissue culture as a fundamental science, it was closely linked with the discovery and characterization of plant hormones and has facilitated our understanding of plant and development. Furthermore, the ability to grow plant cells and tissues in culture and to control their development forms the basis of many practical applications in agriculture, horticulture, phytochemistry and also a prerequisite for plant genetic engineering [1]. Plants have been the basis of many traditional medicine systems throughout the world for thousands of years and it continue to provide mankind with new remedies. Plant based medicines initially dispensed in the form of crude drugs such as herbal formulations, now it could serve as the basis of novel drug discovery. The use of plants as medicines has involved to the isolation of active compounds [2]. Isolation and characterization of pharmacologically active compounds from medicinal plants are being continuously increased in nowadays. Hemigraphis alternata (Burm.f.) T. Ander. belong to the family Acanthaceae. It is an exotic plant adapted to India is an excellent garden plant. In folk medicine, the leaves of H. alternata are ground in to a paste and it is applied on fresh cut wounds. Because it has very good wound healing activity. More over the wound healing property of this potential herb was also proved experimentally [3]. Plant tissue culture helps in providing a basic understanding of physical and chemical requirements of cell, tissues, organs and their and development. The establishment of cell, tissue and organ culture and regeneration of plantlets under in vitro conditions has opened up new avenues in the area of plant biotechnology. In addition to that, such In vitro studies also lead to reveal active principles behind various potential medicinal herbs [4]. Materials and methods Classification and Description of the Plant Kingdom: Plantae Class: Dicotyledons Sub class: Gamopetalae ~ 247 ~

2 Order: Lamiales Family: Acanthaceae Genus: Hemigraphis Species: Hemigraphis alternata (Burm.f.) T. Ander. Synonyms: Ruellia alternata Burm.f. Hemigraphis colorata Hall. Fl.&Fr.: Feb. May Habitat: Grown as garden plant Distribution: Native of Central America Local Name: Murikootti Description of the Plant: It is a prostrate tropical perennial herb and it typically grows to 6-9 in. tall and spreads indefinitely along the ground rooting at the stem nodes as it goes. It is primarily valued as a ground cover for its colourful foliage. Leaves are ovate-cordate leaves toothed, puckered (to 3 in long) are metallic silvery graygreen above and purple beneath. Inflorescence is raceme in which tiny, five-lobed, bell-shaped, white flowers. Flowers are insignificant (Fig.1). Fig.1: Images of Hemigraphis alternata (Burm.f.) T. Ander. Preparation of nutrient medium For the in vitro regeneration, the nutritional media which contains macro and micro elements, sucrose, vitamins, amino acids and regulators. Agar is used as the gelling agent for the culture. In the present study M.S. medium was used (Table-1) [5]. For the preparation of the stock solution, the chemicals such as sucrose 3%, CaCl 2, regulators, agar 0.8%, etc were weighed accurately and mixed by adding required amount of double distilled water. Finally the ph is adjusted to (1 normal NAOH and HCL were used for adjusting the ph). The solution is then subjected to water bath at 80 0 C for about 20 minutes with stirring. Then the medium is transferred into the pre sterilized culturing tubes before solidify. It was then autoclaved (20 minutes, 15 psi pressures at C). The autoclaved tubes containing the medium are kept to solidify and cool before inoculation. The materials like blades, scalpel, conical flask, beakers, Petri dishes, etc used for this process were surface sterilized by treating with detergent and water. It was then rinsed with double distilled water about 3-4 times and autoclaved it for 30 minutes after covering all with aluminum foil. Table 1: M.S. medium composition Constituents Chemical Formula Amount (mg/l) Macronutrients NH₄NO₃ Ammonium nitrate KNO₃ Potassium nitrate CaCl₂.7H₂O Calcium chloride MgSO₄.7H₂O Magnesium sulphate KH₂PO₄ Potassium dihydrogen phosphate Micronutrients Manganese sulphate Zinc sulphate Boric acid Potassium iodide Sodium molybdate Copper sulphate Cobalt chloride Iron Disodium ethylene diamine Tetracetate Ferrous sulphate Sugar Sucrose MnSO₄.4H₂O ZnSO₄.7H₂O H₃BO₃ KI Na₂MO₄.2H₂O CuSO₄.5H₂O CoCl₂.6H₂O NaEDTA FeSO₄.7H₂O ~ 248 ~

3 Regulators Chemical formula Molecular weight Auxins IBA NAA Cytokinins BA KIN Indole-3 butyric acid(c₁₂h₁₃no₂) Naphthalene Acetic Acid(C₁₂H₁₀O₂) 6-Benzyl-Adenine(C₁₂H₁₁N₁₅) Kinetin(6-furfuryl amino purine) (C₁₀H₉N₅O₅) Explant sterilization Nodes of the plant H. alternata were used as explants for the present study. Healthy and good looking nodes were selected. This was then washed under running tap water to remove the dirt. The plant part then washed in neutral detergent Labolene for 5-10 minutes. The material was then washed 3-4 times in double distilled water to remove all the traces of the detergent. It was then surface sterilized by 1% aqueous mercuric chloride solution for 10 minutes and after decanting the sterilant, explants were washed with sterile double distilled water. Inoculation Inoculation was done in laminar air flow cabinet for creating the aseptic condition. Before the inoculation, the blades, scalpel, forceps, etc were flame sterilized by dipping in the alcohol and subjected to flame. The explant was then placed properly in the nutrient medium by pressing it gently. The rim of the culture tube was also flamed before and after the inoculation to avoid the microbial contamination and covered tightly by pre sterilized nonabsorbent cotton plugs. These were all then transferred to the incubation chamber. The culture tubes were maintained at temperature C with relative humidity 60% and a photoperiod of 16/8 hrs with 1000 lux luminance. Followed by regular observation were made. Results and Discussion For the present study M.S. medium supplemented with varying concentration of regulators are used. The inoculated nodal explants of H. alternata is cultured under aseptic and controlled environmental condition in the tissue culture lab. The in vitro reponse of the plant is observed and noted at regular intervals of time. For the shoot initiation M.S. medium supplemented with different concentration of auxin with cytokinins and cytokinins alone is used and for root initiation M.S medium or ¼ M.S medium alone with auxin and auxin with cytokinins are used. The following 10 different concentration of regulators are used for shoot and root regeneration of the study (Table 2,3&4; Fig.2, 3 & 4). Medium Table-2: In vitro response of shooting in H. alternata on M.S medium with varying regulators In vitro response noticed at various time intervals 5 Days 10 Days 15 Days 20 Days 25 Days 3O Days BA 1.0 Mg/l - Shoot initiation Shoot initiation Shoot BA 2.0 Mg/l - KIN 1.0 Mg/l - Green coloration Bulging of explant KIN 2.0 Mg/l Swelling Shoot initiation KIN 1.0 Mg/l + BA 1.0 Mg/l KIN 2.0 Mg/l + BA 2.0 Mg/l Bulging of explant Shoot initiation multiple shoot - Shoot initiation Shoot - Shoot initiation Shoot initiation Shoot Shoot enlargement Shoot Shoot enlargement Leaf formation formation Branching of shoot Leaf formation Table-3: In vitro response of rooting in H. alternata on M.S medium with varying regulators In vitro response noticed at various time intervals Medium 5 Days 10 Days 15 Days 20 Days 25 Days 3O Days NAA 1.0 Mg/l +BA Brown colouration of - No No No No 6.0 Mg/l the medium NAA 1.0 Mg/l + BA Callus Shoot Shoot initiation Slow Root initiation 4.0 Mg/l formation initiation NAA 1.0 Mg/l + KIN Shoot - Shoot Root initiation 4.0 Mg/l initiation IBA 1.0 Mg/l + KIN Shoot Mutiple shoot - Shoot Root development 2.0 Mg/l initiation Callus Root ⅟₄MS+NAA 0.5 Mg/l Shoot and root root branching Root elongation formation initiation ~ 249 ~

4 NAA 1.0 Mg/l + KIN 4.0 Mg/l KIN 1.0 Mg/l + BA 1.0 Mg/l Fig. 2: Direct shoot regeneration in varying concentrations of regulators KIN 2.0 Mg/l KIN 1.0 Mg/l Fig. 3: regeneration in varying concentrations of regulators IBA 1.0 Mg/l + KIN 2.0 Mg/l ~ 250 ~ ¼ M.S.Medium NAA 0.5mg/ Fig. 4: Root regeneration in varying concentrations of regulators The present study was conducted to standardize micropropagation of potential medicinal plant such as H. alternata Similar reports such as in vitro regeneration of plant species belongs to the family Acanthaceae are Rhazya stricta Decne [6], and Adathoda vasica Nees. [7] and Justicia gendarussa [8]. In this study, the medium is Table 4: Effect of auxin with respect to the Length of roots in H. alternate regulator % of response Length of roots NAA 1.0 mg/l + BA 1.0 mg/l - - NAA 1.0 mg/l + BA 4.0 mg/l NAA 1.0 mg/l + KN 4.0 mg/l IBA 1.0 mg/l + KN 2.0 mg/l (1/4 M.S) NAA 0.2 mg/l supplemented with kinetin gives more number of multiple shoots than other cytokinins. Some similar studies observed that, the micropropagation of an elite Medicinal Plant: Stevia rebaudiana Bert [9], they reported that M.S basal medium supplemented with sucrose (30 g L -1 ), agar (7 g L -1 ) and kinetin (2 mg L -1 ) performed best in multiple

5 shoot proliferation, resulting more than 11 multiple shoots from a single shoot tip explant within 35 days of culture. In the present observation on in vitro regeneration of Hemigraphis alternata in M.S. medium supplemented with the regulator Kinetin at a concentration of 2.0 mg/l is found to be more effective than the other cytokinins. Similar studies have been reported in the case of other plants species like Stevia rebaudiana Bertoni. [10], Sinocalamus latiflora Munro [11] and Cucumis melo L. [12]. It was also found that ¼ M.S. medium is more effective for the root initiation than all other auxins. Similar observations were made in in vitro Propagation of Lychnophora pinaster Mar.(Asteraceae) by Desouza et al., Conclusion From the above studies the in vitro regeneration of Hemigraphis alternata on M.S. medium is supplemented with varying concentration of regulators helps to find out the most suitable concentration for the root and shoot regeneration from the nodal explants. From this experiment it is clear that higher amount of cytokinins and lower auxin induce shoot regeneration and high amount of auxin and lower amount of cytokinins induce root regeneration. From this it is concluded that in H. alternata, the most effective shoot regeneration was found in the M.S. medium supplemented with KIN 2 mg/l. It shows an effective rooting in the ¼ strength M.S. medium supplemented with NAA 0.5 mg/l. large numbers of multiple shoots were also found in the M.S. medium is supplemented with KIN 2 mg/l. The present study is an attempt to standardize various factors which are responsible for micro-propagation of potential medicinal herb Hemigraphis alternata. Further studies are also required to reveal and compare the medicinal efficacy of micro-propagated plants with others. 8. Kumar J., Nino T. & Lourthuraj A. (2012). Regeneration of Justicia gendarussa, Ind. J. Innovat. 1(2): Das A., Gantait S. & Mandal N. (2011). Micropropagation of an Elite Medicinal Plant: Stevia rebaudiana Bert. Int. J. Agricult. Res. 6: Abd Alhady, M.R. (2011). In vitro regeneration studies on Stevia rebaudiana Bertoni. Global J. Biotechnol. Biochem. 6(4): Ling Y.M. & Chin C.W. (1987). Micro-propagation studies on Sinocalamus latiflora Plant Sci. 51: Moreno V., Garcia S.M., Granell I., Garcia S.B. & Roig L.A. (1985). In vitro Micro-propagation studies on Cucumis melo L. Plant Cell Tiss. Organ Cult. 5(2): Disouza A.V., Pinto J.E., Bertolucci S.K. & Correa R.M. (2007). In vitro propagation studies on Lychnophora pinaster Mar. (Asteraceae). Hort. Sci. 42(7): References 1. Shahzad A., Faisal M. & Anis M. (2007). Micro propagation through excised root culture of Clitoria ternatea and comparison between in vitro regenerated plants and seedlings. Ann Appl Biol. 150: Uniyal B. (2003). Utilization of medicinal plants by the rural women of Kulu, Himachal Pradesh, Ind. J. Trad. Knowl. 2(4): Sheu J., Jayakumar T., Chang C., ChenY., Priya S., Ong E., Chiou H. & Elizebeth A.R. (2012). Pharmacological actions of an ethanolic extracts of the leaves Hemigraphis colorata and Clerodendron phlomoides. Clin. Mol. Med. 3: Sharma N., Chandel K.P. & Paul A. (1993). In vitro propagation of Gentiana kurroo: an indigenous threatened plant of medicinal importance. Plant Cell Tiss. Org. Cult. 34: Murashige M. & Skoog F. (1962). A revised medium for rapid and bioassays with tobacco tissue cultures. Plant Physiol. 15: Adel T., Mohamed A., Omar A. & Salman D. (2012). In vitro regeneration of plant species belongs to the family Acanthaceae is Rhazya stricta Decne. J. Med. Plants Res. 6(19): Bimal R. & Shahnawaz M.D. (2011). In vitro regeneration of plant species such as Adathoda vasica Nees. J. Med. Plants Res. 6(7): ~ 251 ~